Most DNA sequencing today is carried out by chain termination methods of DNA sequencing. The most popular chain termination methods of DNA sequencing are variants of the dideoxynucleotide mediated chain termination method of Sanger. See, Sanger et al. (1977) Proc. Nat. Acad. Sci., USA 74:5463-5467. For a simple introduction to dideoxy sequencing, see, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., (e.g., Supplement 38, current through 1998) (Ausubel), Chapter 7. Thousands of laboratories employ dideoxynucleotide chain termination techniques. Commercial kits containing the reagents most typically used for these methods of DNA sequencing are available and widely used. In addition to the Sanger methods of chain termination, new PCR exonuclease digestion methods have also been developed for DNA sequencing. Direct sequencing of PCR generated amplicons by selectively incorporating boronated nuclease resistant nucleotides into the amplicons during PCR and digestion of the amplicons with a nuclease to produce sized template fragments has been performed (Porteret al. (1997) Nucleic Acids Research 25(8):1611-1617). The above methods typically require that the terminated fragments be sequenced upon completion of the reaction. This is a time consuming step that limits the ability to sequence in a high throughput manner.
The development of microfluidic technologies by the inventors and their co-workers has provided a fundamental shift in how artificial biological and chemical processes are performed. In particular, the inventors and their co-workers have provided microfluidic systems that dramatically increase throughput for biological and chemical methods, as well as greatly reducing reagent costs for the methods. In these microfluidic systems, small volumes of fluid are moved through microchannels by electrokinetic or pressure-based mechanisms. Fluids can be mixed, and the results of the mixing experiments determined by monitoring a detectable signal from products of the mixing experiments.
Complete integrated systems with fluid handling, signal detection, sample storage and sample accessing are available. For example, Parce et al. xe2x80x9cHigh Throughput Screening Assay Systems in Microscale Fluidic Devicesxe2x80x9d WO 98/00231 and Knapp et al. xe2x80x9cClosed Loop Biochemical Analyzersxe2x80x9d (WO 98/45481; PCT/US98/06723) provide pioneering technology for the integration of microfluidics and sample selection and manipulation. For example, in WO 98/45481, microfluidic apparatus, methods and integrated systems are provided for performing a large number of iterative, successive, or parallel fluid manipulations. For example, integrated sequencing systems, apparatus and methods are provided for sequencing nucleic acids. This ability to iteratively sequence a large nucleic acid (or a large number of nucleic acids) provides for increased rates of sequencing, as well as lower sequencing reagent costs. Applications to compound screening, enzyme kinetic determination, nucleic acid hybridization kinetics and many other processes are also described by Knapp et al.
New or improved methods of sequencing are accordingly desirable, particularly those that take advantage of high-throughput, low cost microfluidic systems. The present invention provides these and other features by providing new sequencing methods and high throughput microscale systems for providing sequencing reactions as well as many other features that will be apparent upon complete review of the following disclosure.
The present invention provides novel methods of sequencing by synthesis or incorporation. Nucleotides or nucleotide analogs are added to reaction mixtures comprising nucleic acid templates and primers, e.g., DNA or RNA. The nucleotides are incorporated into the primer, resulting in an extended primer. The sequence is determined as each additional complementary nucleotide is incorporated into the primer and the steps are repeated until the entire template sequence or a portion thereof is determined.
In one embodiment, the nucleotides or nucleotide analogs, or a fraction thereof, comprise a 3xe2x80x2-blocking group and a detectable label moiety, which typically comprises a phosphate or a carbamate group. The 3xe2x80x2-blocking groups provide reversible chain termination. When added to a growing nucleic acid chain, these nucleotide analogs result in a non-extendable primer. The 3xe2x80x2-blocking group is typically removed, e.g., by a reducing agent and/or a phosphatase, to produce an extendable primer to which further nucleotides are added, thereby allowing continued sequencing of the nucleic acid template. Removal of the 3xe2x80x2-blocking group is optionally performed before or after detection of the added nucleotide.
In another embodiment, the nucleotides or nucleotide analogs comprise a fluorescent label. Sequencing by synthesis using fluorescent nucleotides typically involves photobleaching the fluorescent label after detecting an added nucleotide. Photobleaching comprises applying a light pulse that destroys or reduces to an acceptable level, e.g., a background level or to a low enough level to prevent signal buildup over several sequencing cycles, the fluorescence of the nucleotides, e.g., a fluorescent nucleotide that has been added to the primer. The light pulse is typically applied for about 20 seconds or less, about 10 seconds or less, about 2 seconds or less, about 1 second or less, or about 0.1 second or less. The light pulse typically has a wavelength equal to the wavelength of light absorbed by the fluorescently labeled nucleotides. Detection of the added fluorescently labeled nucleotide occurs prior to or concurrent with photobleaching of the fluorescently labeled nucleotides and/or the extended primer. Nucleic acid templates comprising about 50 or more nucleotides, about 100 or more nucleotides, about 500 or more nucleotides, about 1000 or more nucleotides, about 2000 or more nucleotides, or about 10,000 or more nucleotides are optionally sequenced using these methods, e.g., sequenced with at least about 80%, at least about 90%, or at least about 95% accuracy.
In another embodiment, sequencing comprises sequencing by synthesis using detection of intercalating dyes (xe2x80x9csequencing by intercalationxe2x80x9d). An intercalating dye is incubated or mixed with the template and primer as the sequencing reactions occur. When a nucleotide, e.g., a naturally occurring, non-labeled nucleotide, is incorporated into the primer, it forms an extended double-stranded region, into which intercalating dyes insert, e.g., between the stacked bases. The intercalating dye is detected, thus detecting the addition of a nucleotide to the growing chain and sequencing the template nucleic acid. The intercalating dye optionally comprises ethidium, ethidium bromide, an acridine dye, an intercalating nucleic acid stain, a cyanine dye, such as SYBR green, proflavin, propidium iodide, acriflavin, proflavin, actinomycin, anthracyclines, or nogalamycin. In some embodiments, photobleaching is performed after detecting the intercalating dye or approximately concurrent with detecting the intercalating dye.
The nucleotides or nucleotide analogs in the present invention typically comprise nucleoside 5xe2x80x2-triphosphates (dNTPs), e.g., deoxyadenosine 5xe2x80x2-triphosphate (dATP), deoxyguanosine 5xe2x80x2-triphosphate (dGTP), deoxycytidine 5xe2x80x2-triphosphate (dCTP), deoxythymidine 5xe2x80x2-triphosphate (dTTP), deoxyuridine 5xe2x80x2-triphosphate (dUTP), adenosine 5xe2x80x2-triphosphate (ATP), guanosine 5xe2x80x2-triphosphate (GTP), cytidine 5xe2x80x2-triphosphate (CTP), uridine 5xe2x80x2-triphosphate (UTP), or analogs thereof.
In some embodiments, the nucleotides or nucleotide analogs, or a fraction thereof, comprise a detectable label moiety, e.g., a fluorescent or chemiluminescent label moiety. Different nucleotides optionally comprise detectably different labels, e.g., ATP, GTP, CTP, TTP, and UTP each optionally comprising a distinguishable label.
The nucleotides are optionally incubated with the nucleic acids in series or in combination. For example, four detectably different nucleotides, e.g., reversible chain terminating nucleotide analogs, are optionally simultaneously incubated with template, primer, and polymerase. The added nucleotide stops chain growth, is detected, and then the chain terminating portion of the nucleotide is removed, e.g., the 3xe2x80x2-blocking group is removed to allow further extension of the primer. For example, the 3xe2x80x2-blocking group is optionally removed in a buffer or wash that also removes all unincorporated nucleotides. Alternatively, the 3xe2x80x2-blocking group comprises the label moiety and is detected after removal.
Alternatively, four nucleotides are added in series, one after the other. For example, one nucleotide is added, unincorporated nucleotides are removed from the reaction, and a fluorescent signal is detected, e.g., from a fluorescently labeled nucleotide added to the primer chain or from an intercalating dye that has intercalated into the recently extended double-stranded nucleic acid region. A second nucleotide is added, a third, and so on, e.g., until the nucleic acid template or a portion thereof is sequenced.
In another aspect, the methods involve performing the sequencing, e.g., by incorporation, by photobleaching, by intercalation, and the like, in a microfluidic device. Nucleic acid templates, e.g., DNA or RNA, and primers, are flowed through a microscale channel and contacted by a polymerase and one or more nucleotides or nucleotide analogs in the microscale channel, thereby adding at least one of the one or more nucleotides or nucleotide analogs to the primer. The added nucleotide or nucleotide analog is detected and the steps are repeated, e.g., to obtain an entire sequence or a portion thereof.
In one embodiment, the template and/or primer are attached to a set of particles, e.g., an ordered array of particles, which is flowed through a microscale channel or positioned, e.g., in a fixed location, within the microscale channel. The sequencing reagents, e.g., a train of reagents, are flowed across the particles to sequence the template nucleic acid. Unincorporated nucleotides or reagents are flowed through the microchannel, e.g., to a waste reservoir. Alternatively, the particle sets are flowed through the train of reagents to perform the sequencing. In some embodiments, the reagents are attached to particle sets and the template is flowed through the particle sets to be sequenced. When a nucleotide or nucleotide analog is added to the primer, a signal is typically detected from the added nucleotide, e.g., on the particle sets or released from the particle set and flowed through a detection region.
The particle sets optionally comprise about 1 or more particles, about 10 or more particles, about 100 or more particles, about 1000 or more particles, or about 10,000 or more particles. In some embodiments, the set of particles comprises a set of beads, which beads are selected from: polymer beads, silica beads, ceramic beads, clay beads, glass beads, magnetic beads, metallic beads, paramagnetic beads, inorganic beads, and organic beads; and wherein the beads have a shape, which shape is selected from one or more of: spherical, helical, cylindrical, spheroid, irregular, rod-shaped, cone-shaped, cubic, and polyhedral.
The train of reagents that is used to perform sequencing of a nucleic acid template typically comprises sequencing reagents for performing sequencing, e.g., sequencing by synthesis, e.g., with detection by photobleaching, by pyrosequencing chemistry, or by intercalation. Typical reagents include, but are not limited to, one or more of: a template, a primer, a polymerase, a sufurylase, an apyrase, an inorganic phosphate, ATP, a thermostable polymerase, luciferin, luciferase, an endonuclease, an exonuclease, Mg++, a molecular crowding agent, a buffer, a dNTP, a salt, a phosphatase, a reducing agent, a modified dNTP, a nucleotide, a nucleotide analog, a nucleotide analog comprising a 3xe2x80x2-blocking group, a nucleotide analog comprising a 3xe2x80x2-phosphate group, a nucleotide analog comprising a 3xe2x80x2-carbamate group, a chain-terminating nucleotide analog, a reversible chain terminating nucleotide analog, a fluorescently labeled nucleotide, and an intercalating dye.
The particles and the reagent train are typically flowed through the microscale channel by one or more of: pressure, centripetal force, centrifugal force, a moving magnetic field, and an electrokinetic force.
Microfluidic devices for sequencing a nucleic acid are also provided. The devices typically comprise a body structure having a microscale cavity disposed therein; and a set of particles, e.g., an ordered array of particles as described above, disposed within the microscale cavity. The set of particles comprises at least one set of nucleic acid templates and at least one set of nucleic acid primers. Nucleotides and/or nucleotide analogs, as described above, are also disposed within the device, e.g., in reservoirs or attached to one or more particle sets.